The Tax oncoprotein of human T cell leukemia virus type 1 constitutively activates transcription factor NF-B by a mechanism involving Tax-induced phosphorylation of IB␣, a labile cytoplasmic inhibitor of NF-B. To trigger this signaling cascade, Tax associates stably with and persistently activates a cellular IB kinase (IKK) containing both catalytic (IKK␣ and IKK␤) and noncatalytic (IKK␥) subunits. We now demonstrate that IKK␥ enables Tax to dock with the IKK␤ catalytic subunit, resulting in chronic IB kinase activation. Mutations in either IKK␥ or Tax that prevent formation of these higher order Tax⅐IKK complexes also interfere with the ability of Tax to induce IKK␤ catalytic function in vivo. Deletion mapping studies indicate that amino acids 1-100 of IKK␥ are required for this Tax targeting function. Together, these findings identify IKK␥ as an adaptor protein that directs the stable formation of pathologic Tax⅐IKK complexes in virally infected T cells.During an adaptive immune response, antigen-stimulated CD4 ϩ T lymphocytes become committed to an activation program that triggers a transient phase of clonal expansion (1). In contrast, infection with human T cell leukemia virus type 1 (HTLV-1) 1 can lead to the loss of cell cycle control and development of an aggressive malignancy called adult T cell leukemia (2). The Tax oncoprotein encoded by HTLV-1 stimulates the constitutive nuclear expression of transcription factor NF-B, which regulates antigen-directed T cell proliferation (3, 4).Studies with Tax-transgenic mice suggest that this viral/host interaction is required to maintain the transformed phenotype of HTLV-1-infected cells (5).In quiescent T cells, the activity of NF-B is controlled from the cytoplasmic compartment by virtue of its signal-dependent interaction with inhibitors, including IB␣ (6). Recent studies have identified two cytokine-inducible IB kinases (IKKs), termed IKK␣ and IKK␤, that target IB␣ for degradation via phosphorylation at Ser-32 and Ser-36 (7). These two kinases form heterodimers and function as catalytic subunits within a 700 -900-kDa multicomponent complex (8). Whereas IKK␣ and IKK␤ are activated transiently in cells treated with the cytokine tumor necrosis factor-␣ (TNF) (8 -10), Tax induces their constitutive expression in HTLV-1-infected T cells (11,12). We have recently found that Tax-induced activation of both IKK and NF-B requires the formation of Tax⅐IKK complexes (12). However, the precise mechanism of Tax action on IKKs remains unclear.Here we provide several lines of experimental evidence indicating that Tax-directed IKK activation is mediated by IKK␥ (also called NEMO, IKKAP1, or FIP-3), a recently identified subunit of TNF-responsive IKKs whose precise signaling function is unknown (13-16). First, interference with IKK␥ expression in T cell transfectants inhibits Tax-mediated activation of NF-B. Second, IKK␥ and Tax interact stably in the context of a high molecular mass IB kinase derived from HTLV-1-infected T cells. Third, overexpression of IKK␥ in vivo is suffic...

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV1) chronically activates transcription factor NF-B by a mechanism involving degradation of IB␣, an NF-B-associated cytoplasmic inhibitor. Taxinduced breakdown of IB␣ requires phosphorylation of the inhibitor at Ser-32 and Ser-36, which is also a prerequisite for the transient activation of NF-B in cytokine-treated T lymphocytes. However, it remained unclear how Tax interfaces with the cellular NF-B/IB signaling machinery to generate a chronic rather than a transient NF-B response. We now demonstrate that Tax associates with cytokine-inducible IB kinase (IKK) complexes containing catalytic subunits IKK␣ and IKK␤, which mediate phosphorylation of IB␣ at Ser-32 and Ser-36. Unlike their transiently activated counterparts in cytokine-treated cells, Tax-associated forms of IKK are constitutively active in either Tax transfectants or HTLV1-infected T lymphocytes. Moreover, point mutations in Tax that ablate its IKK-binding function also prevent Tax-mediated activation of IKK and NF-B. Together, these findings suggest that the persistent activation of NF-B in HTLV1-infected T-cells is mediated by a direct Tax/IKK coupling mechanism.The NF-B/Rel family of transcription factors plays an important regulatory role in T-cell homeostasis and antigendriven proliferation (1-4). In quiescent T lymphocytes, NF-B is trapped in the cytoplasm by various inhibitory proteins, including IB␣ (5). In response to mitogenic signals, IB␣ is targeted for destruction by the ubiquitin-proteasome pathway and then NF-B translocates to the nucleus (6). Proteolytic inactivation of IB␣ requires signal-dependent phosphorylation of the inhibitor at Ser-32 and Ser-36 (7-10). Recent studies have identified two cytokine-inducible IB kinases (IKKs) 1 , termed IKK␣ and IKK␤, which appear to form heterodimers and catalyze these site-specific modifications in the context of a multisubunit enzyme complex (11)(12)(13)(14)(15).Human T-cell leukemia virus type 1 (HTLV1) is the etiologic agent of an aggressive malignancy of activated CD4 ϩ T lymphocytes (16). The HTLV1 provirus encodes a 40-kDa oncoprotein, termed Tax, which potently induces the constitutive nuclear expression of . Studies with Tax-transgenic mice suggest that this viral/host interaction is required to maintain the transformed phenotype of HTLV1-infected cells (20). We demonstrated recently that Tax converts IB␣ into a labile proteasome substrate by a mechanism involving phosphorylation of the inhibitor at 21,22). These findings suggested that Tax induces a chronic NF-B response by acting upstream of one or more IKKs (7). However, the precise mechanism by which Tax accesses the host NF-B signaling pathway remained unknown.We demonstrate here that cytokine-inducible IB kinases containing IKK␣ and IKK␤, which normally function in a transient manner (12-14), are expressed as constitutively active signal transducers in HTLV1-infected T lymphocytes. These activated forms of IKK associate stably with Tax when the oncoprotein is expressed i...

The NF-B/Rel family of transcription factors plays a critical role in the development of a normal immune response (39, 65, 75). The nuclear activities of NF-B/Rel proteins are regulated from the cytoplasmic compartment by interactions with a set of inhibitory proteins, including IB␣ and IB␤ (26,71,74). Cellular stimulation leads to the rapid degradation of these conditionally labile inhibitors and the transit of active NF-B/Rel complexes to the nucleus (9,26,74). Primary structural and functional analyses have indicated that IB␣ and IB␤ are organized as tripartite molecules containing (i) an N-terminal regulatory domain required for induced proteolytic breakdown (15,16,18,21,49,62,68,73,76), (ii) a central Rel-interactive domain composed of six ankyrin repeat motifs (25,33,35), and (iii) a highly acidic C-terminal region which is rich in proline, glutamic acid, serine, and threonine residues (30, 72). Sequences of the latter type, termed PEST domains, have been implicated as cis-acting proteolytic signals that determine the metabolic turnover rates of specific proteins (56, 57). However, the function of the PEST domains in IB proteins remains controversial. In contrast to prior reports (16, 76), recent mutational studies have suggested that the PEST domain of IB␣ is dispensable for its signal-induced turnover in activated cells (68). Other experiments have indicated that phosphorylation of the IB␣ PEST domain by casein kinase II regulates basal rather than induced turnover of this inhibitor (46,48,64).

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